The present invention pertains to improved methods for detecting nucleic acid sequences in biological samples, particularly sequences derived from infectious microorganisms.
Hepatitis C Virus (HCV) is a parenterally transmitted virus responsible for the majority of cases of post-transfusion hepatitis and a substantial portion of sporadic (or community acquired) hepatitis cases worldwide. It is estimated that more than 1% of the world""s population is infected with HCV. HCV infection is associated with acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma.
HCV is currently classified as a separate genus, Hepacivirus, in the family Flaviviridae. Its genome consists of a positive-stranded RNA molecule of about 9,500 nucleotides with a single, large open reading frame (ORF) which encodes a polyprotein precursor of about 3,000 amino acids. The large ORF is preceded by a 5xe2x80x2 non-coding region (NCR) of about 340 nucleotides, which is the most highly conserved region of the genome. The 5xe2x80x2 region of the ORF encodes (in a 5xe2x80x2-to-3xe2x80x2 direction) a capsid protein, two envelope glycoproteins (E1 and E2), and a small protein of unknown function (P7). The 3xe2x80x2 portion of the ORF encodes nonstructural proteins which include a protease, protease/helicase bi-functional protein, RNA polymerase, and regulatory peptides. The 3xe2x80x2 portion also includes an NCR.
Analysis of HCV coding sequences from around the world has revealed considerable sequence variation among individual viral isolates. Furthermore, analyses of HCV sequences from individual patients have shown that the virus circulates as so-called xe2x80x9cquasi-species,xe2x80x9d which contain related but not identical sequences. The variation that exists among isolates and within individual patients is believed to be the result of the low fidelity of the virally-encoded RNA-dependent RNA polymerase. The degree of genetic variability of HCV has important implications for prevention, diagnosis, and control of infection.
Serodiagnosis of HCV infection is typically determined by commercially available enzyme immuno-assays (EIA) which detect antibodies that bind recombinant HCV proteins or peptides. Positive EIA results can be confirmed by a recombinant immunoblot assay (RIBA), but neither EIA nor RIBA assays distinguish past from present infections. Because of the typically low titer of circulating virus, a direct assay for viral proteins has not been successfully developed. Furthermore, antibody-based assays fail to detect HCV infection for usually 2 to 3 months after exposure
Thus, there is a need in the art for improved assays for HCV that are sensitive enough to detect HCV viremia within a few days after initial exposure of a patient to HCV.
Thus, in a first aspect, the invention is directed to a method for detecting the presence of Hepatitis C Virus (HCV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using as a template RNA derived from the sample to produce HCV-specific reverse transcription products;
(B) amplifying the reverse-transcription products using one or more pairs of oligonucleotide primers specific for HCV to produce HCV-specific amplification products,
xe2x80x83where each of the pairs comprises:
(a) a forward primer selected from the group consisting of:
(b) a reverse primer selected from the group consisting of:
(C) detecting the amplification products,
xe2x80x83where detection of the amplification products indicates the presence of HCV RNA in the sample.
In another aspect, the invention is directed to a method for amplifying Hepatitis C Virus (HCV) DNA. The method comprises:
(A) performing a polymerase chain reaction on a DNA sample containing HCV DNA using one or more pairs of oligonucleotide primers specific for HCV to produce HCV-specific amplification products,
xe2x80x83where each of the pairs comprises:
(a) a forward primer selected from the group consisting of:
(b) a reverse primer selected from the group consisting of:
In a third aspect, the invention is directed to a method for detecting the presence of Hepatitis C Virus (HCV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using as a template RNA derived from the sample to produce HCV-specific reverse transcription products;
(B) amplifying the reverse-transcription products using a forward primer and a reverse primer to produce HCV-specific amplification products,
xe2x80x83where the forward primer consists of the oligonucleotide 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACG-3xe2x80x2 (1F27)  less than SEQ ID NO. 8 greater than  and the reverse primer consists of the oligonucleotide 5xe2x80x2-AGGCCAGTATCAGCACTCTCTGCAGTC-3xe2x80x2 (57R27)  less than SEQ ID NO. 9 greater than ; and
(C) detecting the amplification products,
xe2x80x83where detection of the amplification products indicates the presence of HCV RNA in the sample.
In a fourth aspect, the invention is directed to a method for amplifying Hepatitis C Virus (HCV) DNA. The method comprises:
(A) performing a polymerase chain reaction on a DNA sample containing HCV DNA using a forward primer and a reverse primer to produce HCV-specific amplification products,
xe2x80x83where the forward primer consists of the oligonucleotide 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACG-3xe2x80x2 (1F27)  less than SEQ ID NO. 8 greater than  and the reverse primer consists of the oligonucleotide 5xe2x80x2-AGGCCAGTATCAGCACTCTCTGCAGTC-3xe2x80x2 (57R27)  less than SEQ ID NO. 9 greater than .
In a fifth aspect, the invention is directed to a method for detecting the presence of Hepatitis C Virus (HCV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using as a template RNA derived from the sample to produce HCV-specific reverse transcription products;
(B) amplifying the reverse-transcription products using one or more pairs of 5xe2x80x2 NCR oligonucleotide primers specific for HCV and one or more pairs of 3xe2x80x2 NCR oligonucleotide primers to produce HCV-specific amplification products,
xe2x80x83where each of the pairs of 5xe2x80x2 NCR oligonucleotide primers comprises:
(a) a forward primer selected from the group consisting of:
(b) a reverse primer selected from the group consisting of:
xe2x80x83where each of the pairs of 3xe2x80x2 NCR oligonucleotide primers comprises a forward primer consisting of the oligonucleotide 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACG-3xe2x80x2 (1F27)  less than SEQ ID NO. 8 greater than  and a reverse primer consisting of the oligonucleotide 5xe2x80x2-AGGCCAGTATCAGCACTCTCTGCAGTC-3 (57R27)  less than SEQ ID NO. 9 greater than ; and
(c) detecting the amplification products,
xe2x80x83where detection of the amplification products indicates the presence of HCV RNA in the sample.
In a sixth aspect, the invention is directed to a method for amplifying Hepatitis C Virus (HCV) DNA. The method comprises:
(A) performing a polymerase chain reaction on a DNA sample containing HCV DNA using one or more pairs of 5xe2x80x2 NCR oligonucleotide primers specific for HCV and one or more pairs of 3xe2x80x2 NCR oligonucleotide primers to produce HCV-specific amplification products,
xe2x80x83where each of the pairs of 5xe2x80x2 NCR oligonucleotide primers comprises:
(a) a forward primer selected from the group consisting of:
(b) a reverse primer selected from the group consisting of:
xe2x80x83where each of the pairs of 3xe2x80x2 NCR oligonucleotide primers comprises a forward primer consisting of the oligonucleotide 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACG-3xe2x80x2 (1F27)  less than SEQ ID NO. 8 greater than  and a reverse primer consisting of the oligonucleotide 5xe2x80x2-AGGCCAGTATCAGCACTCTCTGCAGTC-3 (57R27)  less than SEQ ID NO. 9 greater than .
In some embodiments of the method, where a reverse transcription reaction is performed, the reverse transcription reaction is performed using random oligonucleotide primers, alternatively, one or more HCV-specific reverse transcription primers, i.e., oligonucleotides having sequences that correspond to sequences in HCV RNA, may be used. Methods for detection of amplification include, without limitation, (a) electrophoresis and (b) capture of amplification products on a solid support to which HCV-specific probes are attached followed by quantifying the bound products using a colorimetric assay.
In a further aspect, the invention is directed to a kit for amplifying HCV DNA derived from HCV RNA. The kit comprises one or more pairs of 5xe2x80x2 NCR oligonucleotide primers, where each of the pairs of 5xe2x80x2 NCR oligonucleotide primers comprises:
(a) a forward primer selected from the group consisting of:
(b) a reverse primer selected from the group consisting of:
In a further aspect, the invention is directed to a kit for amplifying HCV cDNA derived from HCV RNA. The kit comprises one or more pairs of 3xe2x80x2 NCR oligonucleotide primers, where each of the pairs of 3xe2x80x2 NCR oligonucleotide primers comprises a forward primer consisting of the oligonucleotide 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACG-3xe2x80x2 (1F27)  less than SEQ ID NO. 8 greater than  and a reverse primer consisting of the oligonucleotide 5xe2x80x2-AGGCCAGTATCAGCACTCTCTGCAGTC-3 (57R27)  less than SEQ ID NO. 9 greater than .
In an alternate aspect, the invention is directed to a kit for detecting the presence of HCV DNA. The kit comprises one or more pairs of 5xe2x80x2 NCR oligonucleotide primers, where each of the pairs of 5xe2x80x2 NCR oligonucleotide primers comprises:
a forward primer selected from the group consisting of:
(b) a reverse primer selected from the group consisting of:
In yet another aspect, the invention is directed to a kit for detecting the presence of HCV RNA. The kit comprises one or more pairs of 3xe2x80x2 NCR oligonucleotide primers, where each of the pairs of 3xe2x80x2 NCR oligonucleotide primers comprises a forward primer consisting of the oligonucleotide 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACG-3xe2x80x2 (1F27)  less than SEQ ID NO. 8 greater than  and a reverse primer consisting of the oligonucleotide 5xe2x80x2-AGGCCAGTATCAGCACTCTCTGCAGTC-3 (57R27)  less than SEQ ID NO. 9 greater than .
The present inventors have discovered that detection of Hepatitis C Virus (HCV) RNA in biological samples is more efficient when oligonucleotides having sequences complementary to certain sequences present in HCV RNA are used as primers for amplification. The present invention thus provides an improved single-round, reverse transcription/amplification assay which detects low copy levels of HCV RNA. Oligonucleotide primers are selected based on theoretical considerations of sequence conservation, intra- and inter-molecular interactions, and the predicted secondary structures of the amplicon and surrounding sequence. Furthermore, the primers and assay system are designed to allow the co-amplification (and co-detection) of multiple regions of the HCV genome, multiple viral species, and an internal positive control (IPC) RNA (or DNA). Simultaneous amplification/detection of multiple regions of the HCV genome increases assay sensitivity and the co-amplification of an IPC decreases the likelihood of false negative results because of PCR inhibition.
Many techniques in molecular biology, microbiology, recombinant DNA, and protein biochemistry are used in practicing the present invention, such as those explained in, for example, Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M. Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed.); Transcription and Translation, 1984 (Hames and Higgins eds.); A Practical Guide to Molecular Cloning, the series, Methods in Enzymology (Academic Press, Inc.); and Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.).
xe2x80x9cNucleic acidxe2x80x9d or xe2x80x9cpolynucleotidexe2x80x9d as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, such as, for example, DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as xe2x80x9cprotein nucleic acidsxe2x80x9d (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
A xe2x80x9ccomplementxe2x80x9d of a nucleic acid sequence as used herein refers to the antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
A xe2x80x9cprimerxe2x80x9d as used herein is an isolated oligonucleotide between about 10 and about 50 nucleotides in length, preferably between about 12 and about 25 nucleotides in length and most preferably between about 12 and about 18 nucleotides in length, that forms a duplex with a single-stranded nucleic acid sequence of interest and allows polymerization of a complementary strand using, e.g., reverse transcriptase or DNA polymerase.
An xe2x80x9cisolatedxe2x80x9d nucleic acid as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring or a reaction mixture if it is synthetic). An isolated nucleic acid typically contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the components with which it was originally associated.
A nucleic acid sequence that is xe2x80x9cderived fromxe2x80x9d a designated sequence refers to a sequence that corresponds to a region of the designated sequence. This encompasses sequences that are homologous or complementary to the sequence.
An internal positive control (IPC) target nucleic acid refers to a synthetic nucleic acid sequence cloned into a plasmid vector which is subsequently linearized, typically by the action of a restriction endonuclease. An IPC will typically have multiple primer binding sequences surrounding a generic probe-binding region, and acts as a generic control against false negative results in nucleic acid amplification reactions.
The sequence of a preferred internal positive control target DNA is:
Nucleic acids comprising any of the sequences disclosed herein or subsequences thereof can be prepared by conventional methods. For example, DNA can be chemically synthesized using, e.g., the phosphoramidite solid support method of Matteucci et al., 1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al., 1989, J. Biol. Chem. 764:17078, or other well known methods. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, xe2x80x9ccapsxe2x80x9d, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. PNAs are also encompassed by the term xe2x80x9cnucleic acidxe2x80x9d. The nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
Amplification as used herein refers to an iterative process by which a nucleic acid is copied. Suitable methods for amplification include without limitation polymerase chain reaction, ligase chain reaction, strand displacement amplification, transcription mediated amplification, and nucleic acid single base amplification.
The present invention provides methods for detection of HCV in biological samples. The methods are carried out by
(i) performing a reverse transcription reaction using as a template RNA contained within or derived from the sample;
(ii) amplifying the reverse-transcription products using pairs of amplification primers having sequences corresponding to sequences within the 5xe2x80x2 or 3xe2x80x2 non-coding region of HCV RNA, to produce HCV-specific amplification products; and
(iii) detecting HCV-specific amplification products.
Detection of HCV-specific amplification products indicates the presence of HCV RNA in the sample.
According to the invention, a biological sample is obtained from a patient by any conventional means. Suitable biological samples include, without limitation, blood, serum, plasma, urine, breast milk, and cerebrospinal fluid. Preferably, plasma is used as the source of HCV RNA.
The biological sample is treated in any manner that provides access of the reverse transcription reagents to RNA, specifically HCV RNA, contained within the sample. RNA xe2x80x9cderived fromxe2x80x9d a biological sample is any RNA which was originally present in the sample and to which access has been gained by treating the sample. Preferably, RNA is extracted from the sample using any method well known in the art, such as, e.g., methods employing guanidinium thiocyanate, or using commercially available reagents and methods such as, e.g., PureScript(copyright) from Gentra Systems, Inc. (Minneapolis Minn.). Any extraction procedure may be used that results in separation of the RNA from RNases, other proteins, and/or any other components that might interfere with reverse transcription.
The sample is then subjected to reverse transcription using (a) random primers, such as random hexamer primers obtainable from Pharmacia Biotech, Piscataway, N.J. and/or (b) primers derived from the 5xe2x80x2 or 3xe2x80x2 NCRs of the HCV RNA genomic sequence. Reverse transcription is carried out using conventional procedures, such as are described in Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M. Ausubel ed.); in U.S. Pat. No. 5,322,770; in Young, et al., J. Clin. Microbiol. 31(4):882 (1993); Myers et al., Biochemistry 30(3):7661 (1991). Other primers suitable for use as reverse transcription primers, and methods for reverse transcription which can be used in the present invention include those described in copending U.S. patent application Serial No. 60/118,520.
Following the reverse transcription reaction, the products are amplified. Any method for amplification may be used, including, without limitation, polymerase chain reaction (PCR), ligase chain reaction, strand displacement reaction, nucleic acid single base amplification, and transcription mediated amplification. Preferably, PCR is used. Typically, a reaction mixture containing all of the necessary components for PCR is added directly to the reverse transcription reaction mixture. Amplification is then carried out using conditions specified by the primer pairs that are used.
The present inventors have discovered certain pairs of HCV-specific amplification primers are particularly advantageous in detecting HCV RNA in patient samples. Non-limiting examples of useful primers derived from the 5xe2x80x2 NCR of the HCV genome include those listed in Table 1 below.
Any combination of sense and antisense oligonucleotides may be used as forward and reverse amplification primers, respectively. Preferred pairs of 5xe2x80x2 NCR primers for use in amplification include:
In one preferred embodiment, the pair of amplification primers consists of 5xe2x80x2-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3xe2x80x2  less than SEQ ID NO. 1 greater than  and 5xe2x80x2-CGGTTCCGCAGACCACTATGGCTCTC-3xe2x80x2  less than SEQ ID NO. 4 greater than . In another preferred embodiment, the pair of amplification primers consists of 5xe2x80x2-GGGAGAGCCATAGTGGTCTGCGGAA-3xe2x80x2  less than SEQ ID NO. 2 greater than  and 5xe2x80x2-CGGGGCACTCGCAAGCACCCTATCA-3xe2x80x2  less than SEQ ID NO. 7 greater than .
Non-limiting examples of useful primers derived from the 3xe2x80x2 NCR of the HCV genome include a forward primer consisting of 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACG-3xe2x80x2 (1F27)  less than SEQ ID NO. 8 greater than  and a reverse primer consisting of 5xe2x80x2-AGGCCAGTATCAGCACTCTCTGCAGTC-3xe2x80x2 (57R27)
 less than SEQ ID NO. 9 greater than .
The invention also encompasses multiplex amplification, i.e., simultaneous amplification of different sequences using different sets of primers in the same reaction mixture. A preferred set of primer pairs that can be used simultaneously in a multiplex amplification reaction is:
Following amplification, the amplified products may be detected using any method known in the art, including, without limitation, gel electrophoresis in agarose or acrylamide; non-isotopic colorimetric detection such as the SureCellxe2x96xa1 system (see, e.g., Example 1 below); ECi detection; fluorescence, and chemiluminescence. Reagents useful in such detection methods are available from, e.g., Molecular Probes, Eugene, Oreg. and Ortho Clinical Diagnostics, Rochester, N.Y.
The detection of HCV-specific amplification products indicates the presence of HCV RNA in the sample. When gel electrophoresis is used, HCV-specific amplification products are confirmed by their size, as predicted by the location in HCV RNA of the sequences corresponding to the amplification primers used in the reaction. Useful HCV-specific 5xe2x80x2 NCR capture probes useful for colorimetric detection include, without limitation, 5xe2x80x2-GGGTCCTGGAGGCTGCACGACACTCAT-3xe2x80x2  less than SEQ ID NO. 11 greater than , 5xe2x80x2-CCTTTCGCGACCCAACACTACTCGGCT-3xe2x80x2  less than SEQ ID NO. 12 greater than , and 5xe2x80x2-TTTCGCGACCCAACACTACTCGGCT-3xe2x80x2  less than SEQ ID NO. 13 greater than . Useful HCV-specific 3xe2x80x2 NCR capture probes include, without limitation, 5xe2x80x2-GCGGCTCACGGACCTTTCACAGCTA-3xe2x80x2  less than SEQ ID NO. 14 greater than  and 5xe2x80x2-ATGCGGCTCACGGACCTTTCACAGC-3xe2x80x2  less than SEQ ID NO. 15 greater than .
Kits for amplifying HCV DNA derived from HCV RNA can be prepared containing one or more pairs of forward and reverse 5xe2x80x2 NCR oligonucleotide primers, where the forward primers may be 5xe2x80x2-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3xe2x80x2 (C69F28)  less than SEQ ID NO. 1 greater than , 5xe2x80x2-GGGAGAGCCATAGTGGTCTGCGGAA-3xe2x80x2 (C131F25)  less than SEQ ID NO. 2 greater than , or 5xe2x80x2-GTGGTCTGCGGAACCGGTGAGTACAC-3 (C143F26)  less than SEQ ID NO. 3 greater than ; and the reverse primers may be 5xe2x80x2-CGGTTCCGCAGACCACTATGGCTCTC-3xe2x80x2 (C133R26)  less than SEQ ID NO. 4 greater than , 5xe2x80x2-GCAAGCACCCTATCAGGCAGTACCACA-3xe2x80x2 (C282R27)  less than SEQ ID NO. 5 greater than , 5xe2x80x2-CACTCGCAAGCACCCTATCAGGCAGTA-3xe2x80x2 (C287R27)  less than SEQ ID NO. 6 greater than , or 5xe2x80x2-CGGGGCACTCGCAAGCACCCTATCA-3xe2x80x2 (C294R25)  less than SEQ ID NO. 7 greater than .
Kits for amplifying HCV DNA derived from HCV RNA can also be prepared containing one or more pairs of forward and reverse 3xe2x80x2 NCR oligonucleotide primers, where the forward primer may be 5xe2x80x2-GGTGGCTCCATCTTAGCCCTAGTCACG-3xe2x80x2 (1F27)  less than SEQ ID NO. 8 greater than  and the reverse primer may be the oligonucleotide 5xe2x80x2-AGGCCAGTATCAGCACTCTCTGCAGTC-3 (57R27)  less than SEQ ID NO. 9 greater than .
Kits for detecting the presence of HCV DNA, which comprise the 5xe2x80x2 and 3xe2x80x2NCR primers described above for amplification of HCV DNA derived from HCV RNA are also encompassed within the scope of the invention.
The kits may additionally comprise reagents and instructions for reverse transcription, amplification, and product detection. For example, the kits can contain reverse transcriptase, deoxynucleotides, thermostable polymerases suitable for DNA amplification reactions, and reagents for labeling and detection of nucleic acids.
The present invention finds use in the diagnosis of HCV infection in patients; in testing the efficacy of anti-HCV therapeutic regimens; and in screening the blood supply for HCV-infected samples.